Method for analyzing a sample of a complex molecule relatively to a reference batch of the same complex molecule

ABSTRACT

The invention concerns a method for analyzing a sample of a complex molecule relatively to a reference batch of the same complex molecule. Said method is characterized in that it consists in breaking up the complex molecule into at least two molecular sub-entities; in determining, on the basis of the atomic sites of said products of the breakup involved in the breakup reactions, the isotope(s) to be analyzed; and in establishing, for at least part of the breakup products, their isotopic profile; and in comparing the isotopic profile of the products of the breakup with the isotopic profile of the raw material(s) previously indexed and/or with the isotopic profile of the reference complex molecule subjected to the same breakup reactions. The invention is useful for detecting counterfeiting in manufacturing processes.

The present invention relates to a process for the analysis of a sampleof a complex molecule relative to a reference batch of the same complexmolecule, so as particularly to determine their degree of similarityand/or the characterization of their process of production.

Counterfeiting complex products has become a veritable scourge, inparticular in the fine chemical, cosmetic and pharmaceutical industries.The detection of counterfeiting of complex products by physico-chemicalanalysis is often based on the analysis of traces of secondary productsof the synthesis, of catalysts or impurities. For example, it has beendetermined by chromatographic analysis in liquid phase (Asakawa,Shuichi; Kato, Koichi; Inoma, SusumuHakodate Customs Laboratory,Hakodate-shi, 040, Japan, Kanzei Chuo Bunsekishoho (1997), 36, 37-43)that certain glyphosphate base herbicides, produced in the United Statesand imported into Japan, infringe Japanese patents. By also using a gasphase chromatographic technique coupled with mass spectrometry, E.Charton, M. Wierer, J. M. Spieser, A. Van Dorsselaer, and G. Rautmann(European Department for the Quality of Medicines, Council of Europe,Strasbourg, F-67029, Pharm. Pharmacol. Commun. (1999), 5(1), 61-66) havebeen able to detect a counterfeit of a medication, somatropine,described in the European pharmacopea, which was in fact a productderived from somatropine of human origin. Conventional chemical methodshave permitted proving that tablets of a narcotic substance,fenethylline, were prepared by copying a German patent (N. Al-Gharablyand A. R. Al-Obaid, College of Pharmacy, Kind Saud University, Riyadh,11451, Saudi Arabia, J. Forensic Sci. Soc. (1994), 34(3), 165-7).Similarly, counterfeiting of antibiotics of the β-lactam series havebeen studied by capillary electrophoresis, at the “National ForensicChemistry Center” of the “U.S. Food and Drug Administration”, 1141Central Parkway, Cincinnati, Ohio, 45202, USA and described in theJournal of Chromatography, A (1994), 674(1-2), 153-63.

These compositional methods are not always effective and they can leadto false positives. Moreover, they cannot be used in all cases becauseof the absence of characteristic tracers.

Another process for the authentication of the origin of a productconstituted by a mixture of organic compounds, is described in Frenchpatent 2.673.291. This process comprises a separative analysis step forthe product by gas phase chromatography, a step of transformation to CO₂by combustion of the compounds of the product, followed by a step ofanalysis by isotopic mass spectrometry so as to measure the enrichmentin C₁₃ of each compound of the mixture before choosing a compound tomark, particularly by modifying the enrichment in C₁₃ of this compoundor by adding similar molecules whose richness in C₁₃ has first beenincreased or decreased. Enrichment by isotopic marking necessary for theauthentication of the origin of a product is a major drawback of thisprocess. Thus, this step requires the manufacturer to modify hisindustrial process to be able to mark and authenticate his products.This requirement is connected to the series of steps used in theanalysis process, these steps being unable to obtain sufficientlydetailed information as to the origin of the products to avoid a markingstep of the product by enrichment.

There is also known, as is described in British Patent 2120007, ananalysis process consisting in fragmenting a molecule by means of anelectron beam in a mass spectrometer chamber with double focusing toobtain metastable ions analyzable by means of said mass spectrometer.However, in this process, the step of fragmentation does not permitobtaining molecular sub-entities, products perfectly stable andisolable, but rather metastable ions of a lifetime of the order ofseveral fractions of a second. Moreover, the nature of the fragments aswell as the molecular site where the cleavage is carried out by theelectron beam of the mass spectrometer, are conditioned by the presenceof the isotope to be determined. These two characteristics of thisprocess distinguish it fundamentally from a process in which a massspectrometer is used for the isotopic ratios.

Correspondingly, more powerful analytic techniques have been developed.Such is the case of the mass spectrometry of isotopic ratios (MSIR).Thus, it is possible to characterize the natural specific isotopicfractionation by Nuclear Magnetic Resonance Specific Natural IsotopeFractionation (NMR-SNIF method) by measuring the isotopic contents atseveral molecular sites (or even al the sites) of a molecule. However,this technique is at present used only for simple molecules that can bedirectly analyzed.

An object of the present invention is to provide a process for theanalysis of complex molecules based on an original methodology usingisotopic techniques in natural abundance.

Another object of the present invention is to provide a process for theanalysis of complex molecules permitting differentiating a batch ofcomplex molecules relative to another batch and establishing aposteriori the history of the process of production of such a complexmolecule without having first modified the process for production ofsuch a complex molecule.

To this end, the invention has for its object a process for the analysisof a sample of a complex molecule relative to a reference batch of thesame complex molecule so as particularly to determine their degree ofsimilarity and/or the characterization of their process of production,characterized in that the complex molecule is cleaved into at least twomolecular sub-entities, in that, if necessary, at least one of thecleavage products is cleaved into at least two new molecularsub-entities and in that this cleavage operation is repeated on at leasta portion of the cleavage products until there are obtained analyzableand isolable molecular sub-entities, and in that there is determined, asa function of the atomic sites of the cleavage products in question, bygenerally chemical cleavage reactions, the isotope or isotopes to bestudied, in that there is established, for at least one portion of thecleavage products, their isotopic profile and in that the isotopicprofile of the cleavage products is compared to the isotopic profile ofthe first materials already cataloged and taking part in the synthesisprocess of the reference complex molecule and/or in the isotopic profileof the cleavage products of the reference complex molecule subjected tothe same cleavage reactions.

The performance of the above steps permits applying such a process to nomatter what complex molecule, without having proceeded to marking,particularly by enrichment in isotopes of the complex molecule to beanalyzed.

According to a particular embodiment of the invention, starting with aselected isotope or isotopes, there is established the isotopic profileof at least a portion of the cleavage products at least by nuclearmagnetic resonance (NMR) for measurement of the specific positionalisotopic content and if desired by mass spectrometry of the isotopicratios (MSIR) for the measurement of the overall isotopic content. It isto be noted that in the two preceding paragraphs, and in what follows,there is meant by isotopic profile the determination of the isotopicabundance at one or several sites of a molecule and not the measurementof the overall isotopic ratio of the whole molecule, which ratio ismeasured by isotopic mass spectrometry.

The invention resides in the following discovery by the inventors. Mostof the organic molecules are obtained by means of a reaction sequencecomprising a number of steps which can often be large when thecomplexity of the molecule increases. Each of these steps ischaracterized by kinetic isotopic effects (and/or thermodynamic effects)which give rise to specific isotopic fractionation, which is to say aselective isotopic marking, at the atomic sites (H, C, N, O . . . )directly implicated in the reaction or located in the immediate vicinityof the reaction sites. It is thus possible to establish a chart ofisotopic distribution of a complex molecule from isotopic profiles ofthe different steps used. The influence of the first materials and ofthe intermediate reagents adapted to be used, is also taken into accountfor establishing the isotopic profile of the molecule based on theindividual profiles of a more or less great number of its constituentfragments.

Authentication takes place as follows:

On a sample of authentic product P₀ constituting the reference complexmolecule, there is carried out a selective cleavage reaction of themolecule into at least two molecular sub-entities P_(-1a) and P_(-1b)that are lighter. The isotopic effects associated with this cleavagereaction are determined. The specific isotopic compositions of P_(-1a)and P_(-1b) are thus unequivocally connected to that of P₀. The specificisotopic parameters of the molecular sites of the fragments are measuredby the SNIF-NMR method (²H, ¹³C, ¹⁵N). A measurement of the overallisotopic content by isotopic mass spectrometry (MSIR) can also becarried out (¹³C, ²H, ¹⁸O, ¹⁵N, ³⁴S). The selection of the isotopes tobe analyzed is done on the basis of reference data and spectroscopiccharacteristics of the fragment. In numerous cases, the SNIF-NMRmeasurement of ²H will suffice for the characterization.

if the P₋₁ fragments have a molecular size incompatible with a directstudy by SNIF-NMR, the analysis sequence is restarted fromP_(-1(a or b)) to P_(-2(a or b)) and so on until molecules generallyused as primary or intermediate materials are obtained for the synthesisin organic industry.

The same study is then carried out, strictly under the same experimentalconditions, on the complex molecule to be analyzed constituted forexample by a product suspected of being a counterfeit or the result ofan illicit patent copying. The comparison of the results obtained in thetwo studies permits establishing an irrefutable conclusion as to theconformity or non-conformity of the product and of the process used.These two steps on the basis of the process suffice to answer thequestion; conforming or non-conforming?

Setting aside the isotopic reaction effects, the isotopic parameters ofthe fragments Pi determined from the above authentication process arerepresentative of relatively simple molecules which are frequentlyintermediates of the industrial synthesis of the medication or of theactive product in question. These parameters therefore constitutereliable indicators of the basic elements used by the producing companyand can be the object of a more rigorous investigation. In the case ofnon-conformity, they permit, with reference to the data on the moleculesof modest size which may already be cataloged, characterizing the originof the primary materials of the counterfeit product. One can thusconclude, not only that the product does not conform but that it hasbeen prepared by such a catalog process or from such cataloged primarymaterial. The analysis process described above thus permits if desiredidentifying the process used in the production of a non-authenticcomplex molecule.

Moreover, the producer who desires ultimately to authenticate hismedication or active product, even if not protected by a patent, canintroduce into his production system one or several syntheticintermediates, corresponding to one or several Pi fragments having anisotopic profile peculiar to themselves. This method creates in effect amarking of the product without the need to add an oxogenous markingelement (as is the case during use of particular compound markers, metaltraces, or products enriched in heavy isotopes such as ¹³C). A specificisotopic footprint can thus be conferred on the synthesis intermediate(molecule of medium size itself synthesized from petroleum derivativesor extracted from vegetable materials, etc.), either by selectinginitial primary materials of a particular and constant isotopic content,or by acting on the isotopic effects associated with the reactions ofpreparation, extraction, purification of the intermediate correspondingto Pi. In the presence of kinetic isotopic effects, a variation of theyield for example can suffice to modify the fractionation and hence theisotopic profile of the synthetic intermediate. By applying the aboveanalysis process to the complex molecule thus elaborated, there will beobtained one or more Pi fragments on which a unique isotopic profile hasbeen conferred. The company will thus have isotopic parameters of one orseveral fragments of its product which will be unique to it. With thisstrategy, the product will not suffer the drawbacks which attach to theaddition of exogenous elements or to the enrichment by isotopic marking.During inspection, the suspect product is studied under the conditionsdescribed above and the interpretation is carried out in the same way bycomparison of the parameters of the fragments of the suspect product andof the reference product. In this case, the control can be simplifiedbecause it suffices to characterize the typical fragment or fragments.With this process, the producer has a practically incontestable methodof characterizing his product and even of characterizing the batches ofit because he need only change the source of a primary material or theconditions of synthesis of a fragment, to give to Pi a typical profile.

In short, in the scope of the analysis process described above, it ispossible, during production of the complex reference molecule which canbe subjected to the same cleavage reactions as the complex molecule tobe analyzed, to select at least one primary material and/or intermediateproduct and/or conditions of synthesis, so as to give to at least one ofthe cleavage products of the reference complex molecule, called Pi, asabove a unique character detectable during analysis without enrichmentby isotopic markers and/or the addition of exogenous materials.

It is to be noted that the fragments or molecular sub-units are obtainedby suitable chemical degradation processes such as are described in theexample of use. The fragments are then separated and purified by varioustechniques, as for example liquid phase chromatography, gaseous phasechromatography or chromatography on silica gel, distillation,recrystallization, etc. The extraction purification protocols are firstcarefully standardized to avoid any uncontrolled isotopic fractionation.

An example of analysis of the process of production of a complexmolecule is described below.

a) Description of the molecule to be analyzed:

By way of illustration of the process, let us consider the case ofsildenafil citrate [VIAGRA, trademark] produced by Pfizer, belonging tothe category of anti-anginal agents of the pyrazolopyrimidinone type.

Sildenafil cirate has the following chemical structure:

C₂₂ H₃₀ N₆ O₄ S, citrate M=474.6

1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl) phenylsulphonyl]-4-methylpiperazinecitrate

This molecule can be cleaved into several molecular fragments bearing acharacteristic isotopic message, called “isotopic synthons”

The reactions of isotopic retro filiation usable are thus as follows:

Occurrence and synthesis methods that can be used for primary materialsP_(-1b), P_(-2b), P_(-4a1), P_(-4ab2) and P_(-4b):

P_(-1b): N-methylpiperazine C₅H₁₂N₂ M=100.16 CAS 109-01-3

P_(-2b): chloro-sulfonic acid SO₃HCl M=116.52 CAS 7790-94-5

P_(-4a1) and P_(-4a2): 1H-pyrazole, 1-methyl, 3-n propyl, 4-amino,5-cyano or acetamido

C₈H₁₂N₄ or C₈H₁₄N₄O

The synthesis of the substituted 1H-pyrazole ring can take place bymeans of a cyclization reaction in hydrazone from ethyl acylacetate andnucleophilic addition of the CN ion to the carbonyl of the cyclichydrazone.

P_(-4b): 2-ethoxy

benzoic acid C₉H₁₀O₃ M=166.18 CAS 134-11-2

The primary materials P-1b, P-2b and P-4b are available commercially butit is interesting to prepare P-4a1 and P-4a2 by means of conventionalsyntheses of the 1H-pyrazole rings. These syntheses generally usesubstituted hydrazines of the R₁NH—NH₂ type and β-dicarboxyl compoundsR₃—CO—CH₂—CO—R₄.

The isotopic contents of the usable primary materials are welldocumented in the literature.

The isotopic ratios R(i) are expressed in deviations δ(i) ‰ relative toan international reference R(ref) by means of the relationship:

δ(i)=((R(i)/R(ref))−1)*10000

²H and ¹⁸O: V. SMOW (Vienna-Standard Mean Ocean Water)

¹³C: V.PDB (Vienna-Pee Dee Belemnite)

¹⁵N: atmospheric nitrogen

³⁴S: CDT, specimen of Troilite extracted from Diablo Canyon (USA)

The benzine rings of fossil origin (petroleum) are characterized byvalues of δ ²H comprised between −20 and −120‰ and the saturated sidechains between 0 and −70. Measurements are carried out by NMR (SNIF-NMR)for the side chains and the overall content by Mass Spectrometry (MSIR).The overall contents in ¹³C measured by MSIR are generally equal to−28.5‰ with a typical variation of the order of 2‰ and the isotopiccontents of ¹³C of the alkylated or functional side chains are measuredby NMR. According to the synthesis process and the origin of the primarymaterial of the side chains, the values δ ¹³C can vary between −5 and−100‰ and thus offer an important characterization potential.

Nitrated molecules of synthetic origin have ¹³C and ¹⁵N values, measuredby MSIR, which are relatively low and equal respectively to −30‰ (1.5)and −20‰ (10) but, in this latter case, the cyclization reactions ofpyrazoles and xanthines lead to substantial impoverishment in heavyisotopes. At this level, it can be considered that the values of δ ¹⁵Nof the CN group or of the CONH₂ group reflect those of the primarymaterials because the introduction into the 1H-pyrazole pattern takesplace without significant isotopic fractionation. The content in ¹⁵N ofthe NH₂ group is all the lower relative to that of the primary material,the lower is the yield of the reaction.

Commercial chlorosulfonic acids are generally made from sulfuric acidwhose ³⁴S content can vary between −25 and +25 □ according to the originof the primary materal (native sulfur, pyrites) and of the process ofproduction. However, once synthesized, the —SO₂— group is an excellentnatural tracer and the content in ³⁴S is determined by MSIR.

Finally, it is interesting to note that the isotopic mapping of citricacid is very well defined and that the origin of the sildenafil citratecan be precisely determined by consideration of the isotopicdistribution in the citrate fragment. Thus, the content of ²H measuredby NMR varies between −40 and −80‰ for biotechnological citric acids butthe values δ¹³C are equal respectively to −11‰ (1) or −25‰ (1)accordingly as the primary material is constituted by a C34 or C3 sugar.The natural citric acids extracted from fruits such as citrus, pineappleor red fruits have δ²H values that are very near to 0‰ (25).

The range of variation which we have shown proves the feasibility of theprocess for production of a medication or active product. A greatpossibility of choice of isotopic values of one or several fragments isoffered to the producing company desiring to carry out a “naturalmarking” of its product.

d) Characterization of the different reaction steps by establishment ofan isotopic fractionation profile:

Step: level-4→level-3

No modification of the ²H and ¹³C contents of the benzene ring isachieved and, in the same way, the δ¹⁸O value of the ethoxy group mustnot vary. The most significant variation is in the NH₂ function ofP(-4a) which is subject to isotopic fractionation ¹⁵N/¹⁴N proportionalto the kinetic effect α of the formation reaction of the amide bond andthe corresponding fractionation is measured by MSIR.

It is to be noted however that the primary material P(-4b), 2-ethoxybenzoic acid, can be naturally and specifically marked without theaddition of enriched molecules, in the following manner:

The O—C₂H₅ group is naturally marked with ²H, ¹³C or ¹⁸O from suitablychosen ethanol molecules. An ethanol synthesis has ²H values equalrespectively to −100 and −160‰ at the two CH₃ and CH₂ sites with ¹³Ccontents of the order of −28 to −31‰ and 18_(O) contents equal to−5-10‰. Moreover, a natural ethanol could have ²H, ¹³C, or ¹⁸O contentsequal respectively to −200 and −400‰ (²H), −11‰ (¹³C) and +7/+10 (¹⁸O).These two types of commercially available ethoxy groups without enrichedaddition, are easily introduced into the o-hydroxybenzoic acid moleculeby means of conventional reactions to form the primary material P(-4b).The isotopic characteristics of this primary material, which become atypical fragment as described above, are present in the final moleculeof sildenafil citrate.

Step: level-3→level -2

In the course of this step, there can be observed by MSIR characteristicvariations of the δ¹⁵N contents of the nitrogen atoms of thepyrimidinone ring

The δ²H and δ¹⁸O values of the NH and C═O cites are not usable becausethey depend on chemical exchanges with the medium.

Step: level-2→level-1

In the course of this reaction step, the benzine ring is sulfonated bymeans of a reaction of the electrophilic substitution type at lowtemperature. The ³⁴S content, measured by MSIR, can be very slightlymodified, but this modification is the less as the sulfonation yield ishigher. No modification is expected for the other isotopomers of P(-1a).

Step: level-1→level 0

The attachment of the piperazine ring of (P-1b) to the sulfonyl group ofP(-1a) can give rise to a slight decrease in ¹⁵N of the piperazinefragment fixed to the sildanefil sulfate. This decrease, which ismeasured by MSIR, can if desired be characterized in the cleavageproduct of sildanefil citrate. The other isotopic contents are notchanged in the course of this step.

What is claimed is:
 1. A process for the analysis of a sample of acomplex molecule relative to a reference batch of the same complexmolecule so as to determine their degree of similarity and/or the natureof their process of production, comprising: cleaving the complexmolecule into at least two molecular sub-entities, in that, ifnecessary, at least one of the cleavage products is cleaved into atleast two new molecular sub-entities and in that this cleavage operationis repeatable on the molecular sub-entities until analyzable andisolable molecular sub-entities are obtained, in that there isdetermined, as a function of the atomic sites of the cleavage productsin question, by generally chemical cleavage reactions, the isotope orisotopes to be studied, in that there is established, for at least aportion of the cleavage products, their isotopic profile at least bynuclear magnetic resonance NMR for the measurement of the specificpositional isotopic content and in that the isotopic profile of thecleavage products is compared to the isotopic profile of primarymaterials already cataloged and taking part in the synthesis process ofthe reference complex molecule and/or in the isotopic profile of thecleavage products of the reference complex molecule subjected to thesame cleavage reactions.
 2. A process according to claim 1, wherein,starting from a selected isotope or isotopes, there is established theisotopic profile of at least a portion of the cleavage products at leastby nuclear magnetic resonance NMR for the measurement of the specificpositional isotopic content and if desired by mass spectrometry, of theisotopic ratios for measuring the overall isotopic content.
 3. Theprocess according to claim 1, wherein, during production of the complexreference molecule before being subjected to the same cleavage reactionsas the complex molecule to be analyzed, there is selected at least oneprimary material and/or an intermediate product and/or materialsynthesis conditions to give to at least one of the cleavage products ofthe reference complex molecule a unique characteristic detectable duringanalysis without enrichment by isotopic marking and/or the addition ofexogenous elements.
 4. A process for the analysis of a sample of acomplex molecule relative to a reference batch of the same complexmolecule, comprising: cleaving said sample complex molecule so thatanalyzable and isolable molecular sub-entities are obtained; determiningan isotope or isotopes of the cleavage product by Nuclear MagneticResonance (NMR); comparing the isotopic profile of the cleavage productsto the isotopic profiles of primary materials already cataloged andtaking part in the synthesis process of the complex molecule of thereference batch and/or comparing the isotopic profile of the cleavageproducts of the sample to the isotopic profile of the reference complexmolecule subjected to the same cleavage reaction; and analyzing resultsof the comparison to determine the degree of similarity of the complexmolecule of the sample relative to the complex molecule of the referencebatch.
 5. A process for the analysis of a sample of a complex moleculerelative to a reference batch of the same complex molecule, comprising:cleaving said sample complex molecule so that analyzable and isolablemolecular sub-entities are obtained; determining an isotope or isotopesof the cleavage product by Nuclear Magnetic Resonance (NMR); comparingthe isotopic profile of the cleavage products to the isotopic profilesof primary materials already cataloged and taking part in the synthesisprocess of the complex molecule of the reference batch and/or comparingthe isotopic profile of the cleavage products of the sample to theisotopic profile of the reference complex molecule subjected to the samecleavage reaction; and analyzing results of the comparison to determinehow the sample complex molecule was synthesized.